1. Field of the Invention
The present invention relates generally to the field of biology. More specifically, the present invention relates to methods of screening for biologically active molecules against arthropods or biologically active molecules that effectively reduce the ability of vector arthropods to transmit their respective pathogen(s).
2. Description of the Related Art
Biomolecules which either directly kill arthropods or reduce their capacity to transmit pathogens have many potential applications. Examples include plant protection from pests by microbial insecticides (Stewart et al., 1996) and the development of vaccines to block the transmission of pathogens or to directly kill their vectors (Billingsley, 1994, Jacobs-Lorena and Lemos, 1995, Willadsen and Billingsley, 1996, Almeida and Billingsley, 1998). The search is ongoing for new (Bowen et al., 1998) and modified (Marzari et al., 1997,. Kasman et al., 1998) biopesticide molecules, as well as genes which cause insect refractoriness to pathogens (Marshall, 1998) and leads to vaccines against blood-feeding arthropods (Billingsley, 1994, Jacobs-Lorena and Lemos, 1995, Willadsen and Billingsley, 1996, Almeida and Billingsley, 1998). However, the laborious process of identifying genes and gene products in recombinant libraries which have in vivo biological activities often limits the progress of such investigations (King et al., 1997).
High-diversity, phage-display expression libraries readily allow the isolation of high affinity human antibody fragments against almost any antigen without any immunization step (Vaughan et al., 1996, Vaughan et al., 1998). Phage display libraries are also capable of generating panels of antibody fragments with wide specificities for different antigens in complex mixtures (Cai and Garen, 1995) and have been recognized as promising tools for the development of anti-vector vaccines (Willadsen and Billingsley, 1996). Furthermore, phage-display libraries are propagated in the common recombinant host Escherichia coli, which is also the most commonly found bacterium in wild A. gambiae and A. funestus (Straif et al., 1998). Existing phage-display recombinant antibody expression systems therefore represent a convenient system for the delivery of recombinant proteins or peptides, and the phagemids which encode them, to the midgut of these important malaria vectors.
The prior art is deficient in effective methods of screening for biomolecules that either kill or reduce the longevity of an arthropod and for biomolecules which reduce or completely inhibit an arthropod""s ability to act as a host vector for a pathogen. The present invention fulfills this long-standing need and desire in the art.
A novel single-insect feeding, testing and recovery (SIFTER) strategy is described for isolating biomolecules (e.g., clones from nucleic acid libraries), wherein the biomolecules exert a desired biological activity in arthropods (e.g., insects). Theoretically, biomolecules causing desired phenotypes in arthropods may be isolated by allowing groups of arthropods to feed on arrays of single biomolecules and recovering active biomolecules from detectably affected individuals.
A model has been developed, and is described herein, in which arrays of single clones from a library of antibody gene constructs were fed to cages of Anopheles gambiae mosquitoes. Clones were fed as a combination of expressed antibody fragments and bacteria containing the phagemids which encode these fragments. The model screening system reduced the burden of rigorously testing individual clones by up to 125-fold.
The SIFTER strategy has applications which go beyond the described model, including screening for clones which kill arthropods or reduce their competence as disease vectors, thereby leading to biopesticides and vaccines for the control of vector-borne diseases. It is proposed that the principles of SIFTER are applicable to screening any library for biological activity in arthropods and/or insects, if the following criteria can be fulfilled: a) individuals sample the arrays representatively and each individual samples only one clone; b) clones are recoverable from the individual; c) desired activities are readily and rapidly identifiable; and d) background levels of false positives for the activity are low.
One object of the present invention is to provide a method of screening, termed single-insect feeding, testing and recovery, for biologically active molecules directed towards arthropods, preferably insects, even more preferably, mosquitoes.
In an embodiment of the present invention, there is provided a method of screening for a biologically active molecule directed towards a species of arthropod, comprising the steps of: (a) feeding an array of elements, each of which contains a different biomolecule or mixture of biomolecules, to a population of a species of arthropod, wherein individual arthropods preferably feed on a single element but has the opportunity to feed upon any element in the array; (b) testing said arthropods for a desired phenotype, wherein said desired phenotype is indicative of one or more biomolecules, aquired by feeding from an element of the array, possessing biological activity towards said species of arthropod; and (c) recovering biomolecule(s) resulting in said desired phenotype from said arthropods displaying said desired phenotype.
In yet another embodiment of the present invention, there is provided a method of screening for a biologically active molecule directed towards the malaria vector mosquito, Anopheles gambiae, comprising the steps of: (a) feeding an array of biomolecules to a population of Anopheles gambiae, wherein individual Anopheles gambiae preferably feed on a single biomolecule; (b) testing said Anopheles gambiae for a desired phenotype, wherein said desired phenotype is indicative of one or more biomolecules possessing biological activity towards said Anopheles gambiae; and (c) recovering said biomolecule(s) resulting in said desired phenotype from the midgut of said Anopheles gambiae displaying said desired phenotype.
Additionally, the above-described methods may further comprise the step of: performing iterative cycles of steps (a), (b) and (c) until the biomolecule(s) are purified. The above-embodied methods may still further comprise the step of: enriching the array of biomolecules for midgut-specific biomolecules prior to the feeding.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.